The QuantiTect Virus Kit is specially designed for highly sensitive detection of up to 4 viral nucleic acid targets by multiplex, real-time one-step RT-PCR using sequence-specific probes. Multiple viral RNA and/or DNA targets plus internal controls can be detected in a single tube without loss of sensitivity. The concentrated 5x master mix enables the addition of higher sample input and contains HotStarTaq Plus DNA Polymerase. The QuantiTect Virus RT Mix contains a unique formulation of Sensiscript Reverse Transcriptase that is optimized for highly sensitive detection of viral RNA. Two kit formats are available: the QuantiTect Virus Kit for cyclers that require ROX dye for fluorescence normalization and the QuantiTect Virus +ROX Vial Kit for other cyclers. For convenience, the master mix can be stored at 2–8°C.

Serial dilutions of bovine viral diarrhea virus (BVDV) RNA, as indicated, were amplified and analyzed using the QuantiTect Virus Kit. The steep sigmoidal curves enable accurate CT value determination, even at low template amounts (10 copies/μl). [A] Detection of BVDV1 RNA. [B] Detection of BVDV2 RNA. NTC: No template control.|Viral RNA was diluted in serial fivefold dilutions and amplified in duplex with an internal control using the QuantiTect Virus Kit. [A] Amplification plot of the viral targets. [B] Amplification plot of the internal control.|The QuantiTect Virus Kit provides a simple procedure for quantitative, multiplex, real-time PCR. In contrast to current methods, the kits eliminate the need for optimization of the concentrations of primers, Mg2+, and Taq DNA polymerase, even for difficult assays (e.g., assays in which the copy number of the target gene is much smaller than that for the reference gene).|[A] NH4+ ions prevent nonspecific primers from annealing to the template. [B] Synthetic factor MP, an innovative PCR additive, increases the local concentration of primers and probes at the template. Together with K+ and other cations, synthetic factor MP stabilizes specifically bound primers and probes, allowing efficient primer extension by HotStarTaq Plus DNA Polymerase.|Viral RNA was diluted in serial fivefold dilutions and amplified in duplex with an internal control using the QuantiTect Virus Kit (see figure "Reliable detection of viral RNA over a wide linear range") or kits from Suppliers AII and I. The QuantiTect Virus Kit provided much higher sensitivity than the other reagents tested, enabling reliable analysis of unknown samples. ND: Not detected after 50 PCR cycles.|Serial tenfold dilutions of RNA standards (in vitro transcribed RNA) were prepared using either QuantiTect Nucleic Acid Dilution Buffer or RNase-free water, as indicated. These dilutions were used as template in one-step RT-PCR either directly (0 test) or after storage for 2 or 4 weeks at -20°C. Using QuantiTect Nucleic Acid Dilution Buffer resulted in lower CT values and improved stability of the standards.|

QuantiTect Nucleic Acid Dilution Buffer, supplied with the kits, stabilizes RNA and DNA standards during dilution and reaction setup and prevents loss of nucleic acids on plastic surfaces, such as tubes or pipet tips. The buffer enables reliable dilution of standards used to quantify viral nucleic acids, giving a wide linear range, from low to high CT values and ensures longer storage of standards without degradation (see figure "Reliable dilution and storage of RNA standards").

Principle

QuantiTect Virus Kits provide highly sensitive detection of viral nucleic acids in single or multiplex assays on the first attempt (see flowchart "QIAGEN multiplex kits"). The optimized master mix ensures that PCR products in a multiplex reaction are amplified with the same efficiency and sensitivity as PCR products in a corresponding single-amplification reaction.

Amplifying control and target genes in the same reaction, instead of separate reactions, increases the reliability of gene quantification by minimizing handling errors. The QuantiTect Virus Buffer contains a balanced combination of K+ and NH4+ ions as well as unique synthetic Factor MP stabilizes, which together promote stable and efficient annealing of primers and probes to the nucleic acid template, enabling high PCR efficiency (see figure "Unique PCR buffer"). In addition, the unique formulation of Sensiscript Reverse Transcriptase ensures highly sensitive reverse transcription of viral RNA, while HotStarTaq Plus DNA Polymerase provides a stringent hot start, preventing the formation of nonspecific products.

Components of the QuantiTect Virus Kit

Kit component

Feature

Benefits

5x QuantiTect Virus Master Mix

Concentrated master mix

Highly concentrated and optimized for sensitive virus detection

Larger volumes of template can be added to the assay for increased sensitivity

Stabilizes RNA and DNA standards during dilution and reaction setup and prevents loss of nucleic acids on plastic surfaces, such as tubes or pipet tips

Procedure

QuantiTect Virus Kits provide highly sensitive real-time PCR analysis of viral nucleic acids (RNA and/or DNA) and internal controls using sequence-specific probes. Reactions can be carried out with or without a reverse-transcription step, enabling flexible design of multiplex assays to detect RNA targets, DNA targets, or both RNA and DNA targets. Follow the protocol in the handbook for fast and reliable results.

Kits are available with or without ROX passive reference dye in the master mix (see table).